The present invention relates to an immunoassay method, an immunoassay device and an immunoassay kit, permitting easy, quick and highly accurate detection of at least two kinds of test substances from verotoxin-producing Escherichia coli, verotoxin and human hemoglobin in a test sample simultaneously on the same base material.
The O157, which is a verotoxin-producing Escherichia coli posing serious problems in recent years, enters the body with foodstuff, which is the main infectious source, and causes the onset of disease after about 4 to 9 days of incubation period. Bloody feces is a symptom seen from the early stages of the infection and, in some cases, hemolytic anemia, renal failure and thrombocytopenia follow due to the action of verotoxin produced by O157. The disease may ultimately progress to cause hemolytic uremic syndrome (HUS).
The operation to detect verotoxin-producing Escherichia coli in foodstuff and patients is extremely complicated and requires many days before results are obtained. However, an immunological assay method has recently enabled a comparatively easy detection of the causative source.
A specific detection method includes a method (trademark EHEC-TEC ELISA TEST SYSTEM, manufactured by Organon Teknika Corp.) for detecting O157 antigen, comprising culturing a food using mTSB (Tripticase Soy Broth Modified) medium and applying an ELISA method (enzyme-linked immunosorbent assay method). For the detection of verotoxin production by Escherichia coli separated from food, a method (trademark, Verotox-F xe2x80x9cSEIKENxe2x80x9d, manufactured by Denka Seiken Co., LTD.) includes culture thereof in a CA-YE medium and detection of verotoxin 1 and verotoxin 2 by latex agglutination test using supernatant as a test sample.
A method for detecting O157 in a test sample from a patient includes use of a latex agglutination test (trademark Escherichia coli O157detection kit xe2x80x9cUNIxe2x80x9d, manufactured by UNIPATH LTD.).
The above-mentioned methods detect O157 and verotoxin as a single test item, which means that they cannot be detected concurrently. In addition, these methods require enrichment before detection. Thus, they are time-consuming and require complicated manipulation.
On the other hand, an immunity chromatography method has recently been drawing attention as a method permitting quick and easy immunoassay. In this method, the following steps are taken. That is, an immobilized phase on which an immunity substance capable of binding with an assay target substance in a test sample is immobilized on a water absorbable base material, and a label phase comprising a labeled immunity substance capable of binding with said assay target substance, in such a manner that the labeled immunity substance can be released from said base material upon contact with water, are set at specific intervals to give a test strip, and a test sample is absorbed from one end on the label phase side of the strip. Then, the labeled immunity substance is released from the label phase, bound with the assay target substance to form a labeled immunity substancexe2x80x94assay target substance complex, and this complex is bound with an immobilized immunity substance at the aforementioned immobilized phase. By assaying the labeled immunity substance bound at the immobilized phase, the assay target substance in the test sample can be assayed.
The label to be used to give the labeled immunity substance is exemplified by colloidal metallic particle, enzyme, fluorescent material, phosphorescent material, coloring material and water dispersible polymer particles bound with or containing enzyme, fluorescent material, phosphorescent material, coloring material and the like. In particular, a labeled immunity substance wherein water dispersible polymer particles colored with a fluorophosphorescent material, coloring material (e.g., dye and pigment) and the like are bound with an immunity substance by physical adsorption, and a labeled immunity substance obtained by binding gold colloidal particle with an immunity substance are widely used due to high determination sensitivity and easiness.
In the present invention, an immunity chromatography method is used to detect verotoxin-producing Escherichia coli which is represented by O157 raising great concerns these days, verotoxin and human hemoglobin associated with intestinal hemorrhage, and an immunoassay method is provided that permits easy, quick, highly accurate and simultaneous detection of these assay target substances.
According to the present invention, it has been found that, in a conventional immunity chromatography method, the use of a labeled immunity substance wherein a second immunity substance capable of binding with said assay target substances has been labeled with colored particles and an immobilized phase comprising at least two first immunity substances capable of specifically binding with at least two kinds of assay target substances selected from verotoxin-producing Escherichia coli, verotoxin and human hemoglobin in a test sample, the first immunity substances being immobilized on different positions on a water-absorbable base material, enables simultaneous detection of plural assay target substances in a test sample.
Thus, the present invention provides the following.
(1) An immunoassay method comprising bringing an immobilized phase comprising, at different positions on a water-absorbable base material, at least two first immunity substances capable of specifically binding with at least two kinds of assay target substances selected from the group consisting of verotoxin-producing Escherichia coli, verotoxin and human hemoglobin contained in a test sample, into contact with a test sample and a liquid containing labeled immunity substances each comprising a second immunity substance that is labeled with colored particles and capable of binding with said assay target substance, thereby to form an assay target substance-labeled immunity substance complex and to bind said complex with respective first immunity substances at the immobilized phase.
(2) The immunoassay method of above (1), wherein the contact is made by flowing a mixture of the test sample and the liquid, so that it is absorbed from one end of the water-absorbable base material, thereby to bind said complex with the first immunity substance.
(3) The immunoassay method of above (1), wherein the contact is made by flowing the test sample, so that it is absorbed from one end of the water-absorbable base material, thereby to bind the assay target substance with the first immunity substance, and then flowing the liquid to allow absorption thereof by the base material, thereby to bind said labeled immunity substance with the assay target substance.
(4) The immunoassay method of above (1), wherein the contact is made by having the test sample absorbed halfway up to the immobilized phase, allowing the liquid to be absorbed from one end of the water-absorbable base material, thereby to form a complex of said labeled immunity substance and the assay target substance, and binding said complex with the first immunity substance at the immobilized phase.
(5) The immunoassay method of above (1), wherein the contact is made by forming a label phase at a position halfway up to the immobilized phase, the label phase comprising the labeled immunity substance in such a manner that the labeled immunity substance can be released from the base material upon contact with water, allowing the test sample to be absorbed from one end of the water-absorbable base material, thereby to form a complex of said labeled immunity substance and the assay target substance, and binding said complex with the first immunity substance at the immobilized phase.
(6) An immunoassay device comprising an immobilized phase comprising plural first immunity substances each capable of specifically binding with an assay target substance immobilized on a water-absorbable base material, and a label phase comprising a labeled immunity substance comprising a second immunity substance that is labeled with colored particles and capable of binding with said assay target substance, in such a manner that the labeled immunity substance can be released from the base material upon contact with water, said immobilized phase comprising at least two first immunity substances capable of specifically binding with at least two kinds of assay target substances selected from the group consisting of verotoxin-producing Escherichia coli, verotoxin and human hemoglobin contained in a test sample, said first immunity substances being immobilized on different positions on the base material.
(7) An immunoassay kit comprising an immobilized phase comprising, on a water-absorbable base material, plural immobilized first immunity substances each capable of specifically binding with an assay target substance, and a liquid containing labeled immunity substances each comprising a second immunity substance that is labeled with colored particles and capable of binding with said assay target substance, said assay target substance being at least two kinds of assay target substances selected from the group consisting of verotoxin-producing Escherichia coli, verotoxin and human hemoglobin.